Tumor necrosis factor‐α polymorphism and risk of primary nephrotic syndrome: A case–control study and meta‐analysis

Abstract Background The current study aims to explore the relationship between tumor necrosis factor‐α (TNF‐α) polymorphism and the risk of primary nephrotic syndrome (PNS). Methods A total of 250 PNS patients were selected for this study, as well as 300 volunteers serving as the control group. TNF‐α polymorphism were assessed using the polymerase chain reaction‐restriction fragment length polymorphism method. In addition, a meta‐analysis was conducted to analyze previously published literature on this topic. Results No significant differences were observed in the genotypes frequency or alleles frequency among the study populations. Meta‐analysis results revealed a positive association between TNF‐α rs1800629 polymorphism and allele contrast in African populations (p = 0), homozygote comparison (p = .007), heterozygote comparison (p = .026), recessive genetic model (p = .011), and dominant genetic model (p = .000). Conclusions TNF‐α rs1800629 polymorphism does not appear to confer any increased risk for PNS.


| INTRODUCTION
Primary nephrotic syndrome (PNS) is a clinical syndrome characterized by the excessive loss of plasma proteins in urine due to increased glomerular basement membrane permeability.The etiology of PNS is not yet fully understood, and its occurrence and development are believed to be influenced by various factors, including heredity, environment, humoral factors, and immune responses. 1PNS primarily involves immune-mediated glomerular injury, with minimal change disease (MCD), focal segmental sclerosis (FSGS), membranous nephropathy (MN), and mesangial proliferative glomerulonephritis (MsPGN) being the main pathological types observed in patients. 2,3hese immune-mediated autoimmune diseases significantly impact human health.
5][6][7] It is closely associated with glomerular injury and has been extensively studied in multiple diseases, including malignancies, [8][9][10] inflammatory diseases, 11,12 autoimmune diseases, 13,14 cardiovascular and cerebrovascular diseases, 15 as well as infectious diseases. 12,16The TNF-α gene polymorphism is known to influence gene transcription and cytokine synthesis.Among these polymorphisms, TNF-α rs1800629 polymorphism has been extensively investigated in various disorders.Furthermore, the association between TNFalpha gene polymorphisms and childhood nephrotic syndrome has been explored. 17This particular SNP has been found to have a significant impact on gene transcription and cytokine synthesis within multiple disease contexts. 18espite comprehensive investigations on TNF-α polymorphisms, including rs1800629, the specific impact of TNF-α rs1800629 polymorphism on the risk of developing PNS remains to be elucidated.
The aim of this study is to contribute to our understanding of the etiology and risk factors associated with PNS by exploring the potential relationship between TNF-α rs1800629 polymorphism and PNS risk using experimental method and meta-analysis.The findings from this research may have significant implications for the prevention and management of PNS and provide valuable insights for future clinical interventions.

| Chemicals and reagents
A list of the main chemicals and reagents used in our research, together with their respective manufacturers, suppliers and product details, is given in Supporting Information.diagnostic criteria of PNS, which included a 24-h urinary protein level greater than 3.5 g/day and serum protein level below 30 g/L.The patients underwent renal biopsy and had not received hormone drugs within 2 weeks before admission.Exclusion criteria were defined as follows: (1) subsequent nephrotic syndrome, (2) co-infection with fungal, bacterial, or mycoplasma infections, (3) presence of malignant tumors including blood diseases and solid tumors, (4) history of tissue or organ injury such as recent trauma, burn, surgery, or myocardial infarction, and ( 5) presence of autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis.

| Participates characteristics
The control group consisted of 300 healthy volunteers who underwent physical examination during the same period.Control participants had normal blood routine, urine routine, liver function, kidney function and did not have glomerular diseases, tumors or other diseases.Informed consent was obtained from all participants.Detailed demographic characteristics such as age, sex, body mass index (BMI), smoking status, and alcohol Xiao et al. 25 Forest plot for the associations between TNF-α rs1800629 polymorphism and PNS risk through allele contrast (A vs. G).CI, confidence interval; OR, odds ratio; PNS, primary nephrotic syndrome; TNF-α, tumor necrosis factor-α.
consumption were recorded for both PNS patients and control participants (Table 1).

| TNF-α polymorphism analysis
Blood samples obtained from all participants were processed according to standard procedures using a DNA extraction kit.The TNF-α rs1800629 polymorphism was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, which has been extensively described in previous studies.DNA sequencing was performed to validate the genotyping results for a total of 550 samples.The primer sequences used for the TNF-α rs1800629 polymorphism analysis were as follows: forward: 5′-GGAGGCAATAGGTTTTGAGGGC CAT-3′; reverse: 5′-CTGCACCTTCTGTCTCGGTTTCT-3′.

| The process of meta-analysis
A comprehensive literature search was conducted in well-established databases, including PubMed and EM-BASE.The search covered articles published from the inception of these databases until December 2022.Search terms included "polymorphism" or "polymorphisms" together with "primary nephrotic syndrome" or "PNS" and "tumor necrosis factor-alpha" or "TNF-alpha."Only studies that met the following criteria were included in the meta-analysis: (a) case-control studies investigating the association of interest, and (b) studies providing data to estimate effect sizes.Studies were excluded if they met any of the following criteria: (a) not being a case-control study involving human subjects, or (b) lacking sufficient data to estimate effect sizes.Two authors independently conducted the search, review, and evaluation of all included studies.Relevant information such as author names, publication year, and Hardy-Weinberg equilibrium (HWE) status were recorded.The Newcastle-Ottawa Scale (NOS) score was used to assess study quality based on these criteria. 18

| Statistical analysis
Statistical analysis was performed using SPSS version 17.0 software.Measurement data were presented as means ± standard deviation (SD), and comparisons Forest plot for the associations between TNF-α rs1800629 polymorphism and PNS risk through homozygote comparison (AA vs. GG).CI, confidence interval; OR, odds ratio; PNS, primary nephrotic syndrome; TNF-α, tumor necrosis factor-α.

| Participants enrolled in the study
Table 1 described the participants' information of PNS patients and healthy controls.No significant difference was found in the above information which was referred in Section 2 (p > .05).

| TNF-α polymorphism
A total of three genotypes (GA, AA, and GG) were found in the present experiment.In the present experiment, we found no noteworthy difference of distribution rate in both allele and genotype.The detailed information was shown in Table 2.The distribution frequency of TNF-α rs1800629 genotype in PNS group showed no significant bias in gender and age.
F I G U R E 4 Forest plot for the associations between TNF-α rs1800629 polymorphism and PNS risk through heterozygosis comparison (GA vs. GG).CI, confidence interval; OR, odds ratio; PNS, primary nephrotic syndrome; TNF-α, tumor necrosis factor-α.
Our study aimed to investigate the association between TNF-α rs1800629 polymorphism and the risk of primary nephrotic syndrome (PNS).However, we did not find a significant association between TNF-α rs1800629 polymorphism and PNS risk in our study.
Neither genotype nor allele frequencies demonstrated a significant influence on the risk of PNS.This finding suggests that this particular polymorphism may not contribute to an increased or decreased risk of developing PNS.Interestingly, our findings contrast with some earlier studies.Jafar et al. concluded that TNF-α rs1800629 polymorphism may affect susceptibility to idiopathic nephrotic syndrome, 29 and Midan et al. 27 also proposed that this polymorphism might influence the risk of idiopathic nephrotic syndrome.These discrepancies are not uncommon in genetic association studies and can be attributed to various factors, including differences in the study population, racial backgrounds, sample sizes, and other confounding variables.In our study, the population characteristics and other variables were different from those of studies conducted in India and Iran.Although we obtain a positive result by meta-analysis, we believe that the positive results are due to factors such as racial differences and geographical differences.There is much evidence for this in the previously reported literature.Therefore, it is not uncommon for the results of meta-analyses to be inconsistent with those of individual case-control studies.
When considering our findings in the context of immunity, inflammation, and disease progression, it is important to note that TNF-α is a key cytokine involved in both immune responses and inflammation. 30It plays a crucial role in regulating immune cell activation, cytokine production, cell proliferation, apoptosis, and tissue repair processes. 31In PNS, immune-mediated glomerular injury is considered a major pathogenic mechanism. 32The balance between pro-inflammatory cytokines, such as TNF-α, and anti-inflammatory cytokines is crucial for maintaining immune homeostasis and preventing excessive inflammation.Disruptions in this F I G U R E 5 Forest plot for the associations between TNF-α rs1800629 polymorphism and PNS risk through recessive genetic model (AA vs. AG/GG).CI, confidence interval; OR, odds ratio; PNS, primary nephrotic syndrome; TNF-α, tumor necrosis factor-α.
balance can contribute to the development and progression of various autoimmune diseases, including PNS.
While our study did not reveal a direct association between TNF-α rs1800629 polymorphism and PNS risk, it is possible that this polymorphism may have an indirect impact on disease progression and severity through its effects on urinary protein levels.Microalbuminuria, considered an early marker of kidney damage, has been associated with increased risk of CKD progression. 33his study holds several strengths and limitations.Firstly, this study is the first to investigate TNF-α rs1800629 polymorphism in PNS patients both domestically and internationally, contributing to the existing literature on this topic.Additionally, the sample size of this study is relatively large, with more than 500 individuals enrolled in the analysis.5][36][37][38][39][40] However, there are certain limitations that must be acknowledged.Although our study design was innovative in exploring TNF-α rs1800629 polymorphism in PNS patients, there is always room for improvement.2][43][44] Furthermore, well-designed multicenter studies with larger sample sizes are needed to further explore this association using methods such as genome-wide association studies and updated metaanalytical approaches.

| CONCLUSION
Our results indicated that TNF-α rs1800629 polymorphism conferred no risk to PNS.
F I G U R E 6 Forest plot for the associations between TNF-α rs1800629 polymorphism and PNS risk through dominate genetic model (AA/GA vs. GG).CI, confidence interval; OR, odds ratio; PNS, primary nephrotic syndrome; TNF-α, tumor necrosis factor-α.
T A B L E 5 Meta-analysis of the TNF-α rs1800629 polymorphism and PNS risk.

Comparison
Population N

Test of association
T A B L E 4 Quality assessment of the six case-control studies according to the Newcastle-Ottawa scale.
A total of 250 patients diagnosed with PNS were recruited from The People's Hospital of Suzhou New District between March 2018 and March 2023.All patients met the The participates characteristics of both PNS group and control group.
T A B L E 1Abbreviations: CI, confidence interval; PNS, primary nephrotic syndrome; OR, odds ratio.aAdjustedforsex and age by logistic regression model.F I G U R E 1 PRISMA 2009 flow diagram.T A B L E 3 Main characteristics of all case-control studies included in meta-analysis.Abbreviations: HB, hospital-based; HWE, Hardy-Weinberg equilibrium; RFLP, restricted fragment length polymorphism; NOS, Newcastle-Ottawa Score; PB, population-based.